Xenotransplantation using pigs as the transplant source has the potential to resolve the growing shortage of human organ donors. The most profound barrier to xenotransplantation is the immunological rejection of the xenograft and the development of relatively non-toxic immunosuppressive or tolerance-inducting regimens is required to justify clinical trials of pig organs. CD47 is ubiquitously expressed and serves as a ligand of signal regulatory protein (SIRP)1, a key inhibitory receptor on phagocytes and APCs. Our pilot data have shown that: 1) pig CD47 cannot functionally interact with mouse or human SIRP1 and the expression of host- type CD47 on a pig hematopoietic cell line markedly inhibits its phagocytosis by xenogeneic (mouse or human) macrophages;2) induction of mixed chimerism neither overcomes the CD47 barrier nor prevents rejection of pig hematopoietic cells by phagocytes;and 3) CD47-SIRP1 interaction is required for preventing host DC activation and inhibiting anti-donor T cell responses in mice after donor specific transfusion (DST). Based on these and other pilot data described in this application, we hypothesize that CD47 incompatibility makes xenogeneic hematopoietic cells highly susceptible to phagocytosis and augments T cell xenoreactivity by promoting APC activation;thus posing a strong barrier to xenotolerance induction by the mixed chimerism and DST approaches. To test our hypothesis, we will pursue three specific aims. Aim 1 is to evaluate the potential of CD47 transgene expression to inhibit phagocytosis of pig hematopoietic stem/progenitor cells (HSC/HPC). We will determine 1) whether mouse CD47-expressing pig HSC/HPC show reduced susceptibility to phagocytosis and improved ability to engraft in pig cytokine transgenic mice;and 2) if human CD47 expression can protect pig HSC/HPC from phagocytosis by human macrophages. In Aim 2, we will further characterize the interaction of pig CD47 with human SIRP1 (binding vs. function). We will determine: 1) if fusion proteins of pig CD47 extracellular domain can bind to human macrophage SIRP1;2) the ability of pig CD47 to bind and signal through human SIRP1 at the cellular level;and 3) the possible role of CD47 MMS (multiply membrane-spanning) domain in determining the species specificity. In Aim 3, we will use CD47 KO mouse models to further understand how CD47-SIRP1 signaling contributes to DST-induced immunosuppression. We will determine whether the engagement of CD47 on DST donor cells with SIRP1 on recipient DCs can promote the induction of regulatory T cells by inhibiting DC activation/maturation, and if CD47 expression on donor cells is critical for the synergistic effect of DST with costimulatory blockade on tolerance induction. These studies are expected to provide insights into the mechanisms of xenoimmune responses, help in evaluating the value of making human CD47 transgenic pigs for improving xenotolerance induction by mixed chimerism and DST, and enlighten future investigations on the potential roles of other immune inhibitory receptors in xenograft rejection and xenoimmune tolerance.